Induction of cell cycle arrest in human breast cancer cells by adiponectin

Abstract

Objective To investigate the inhibitory effect of adiponectin on the human breast carcinoma cell line MCF7 and to explore the potential mechanism. Methods MCF7 cells were grown in RPMI 1640.The expression of adiponectin receptors in MCF7 cells, including adiponectin receptor 1 and adiponectin receptor 2, was detected by the RT-PCR assay.The total cell count and the percentage of cells at different stages of cell cycle were measured after the exposure to human adiponectin at 20 μg/ml or 40 μg/ml for 48 hours.The expression of phospho-AMPK (THr172)/AMPK, phospho-Akt (Ser473)/Akt, cyclin D1 and cyclin E2 was determined by Western blotting.Comparisons between groups were performed using one-way ANOVA. Results (1) MCF7 cells express both adipo-R1 and adipo-R2.(2) Treatment of the cultured MCF7 cells with human adiponectin resulted in a dosage-dependent suppressive effect on the cell proliferation.At 20 μg/ml and 40 μg/ml, human adiponectin inhibited the growth of MCF7 cells by 13.3% and 27.1%, respectively (F=20.438, P<0.05). (3) Adiponectin treatment leads to suppression of cell proliferation, which is primarily due to the significant increase of cell populations at G1/G0 phase (control group 45.1%±0.7%, 20 μg/ml adiponectin group 49.1%±1.3%, 40 μg/ml adiponectin group 50.1%±1.1%, F=17.339, P<0.05), concomitant with these changes, the percentage of MCF7 cells in S phase was decreased (control group 39.9%±1.2%, 20 μg/ml adiponectin group 36.4%±1.5%, 40 μg/ml adiponectin group 36.0%±2.1%, F=9.715, P<0.05). (4) Adiponectin treatment markedly increases the phosphorylation (Thr172) of AMPK in MCF7 cells within 30 min.The expression of phospho-AMPK-α was 0.44±0.01, 0.67±0.02 and 0.72±0.03 in the control group, the 20 μg/ml adiponectin group and the 40 μg/ml adiponectin group, respectively (F=91.07, P<0.05). Prolonged exposure to adiponectin for 24 hours resulted in the expression reduction of cyclin D1.The expression of cylcin D1 was 0.81±0.03, 0.70±0.03 and 0.59±0.03 in the control group, the 20 μg/ml adiponectin group and the 40 μg/ml adiponectin group, respectively (F=30.25, P<0.05). The treatment of adiponectin for 48 hours markedly decreased the expression of cyclin E2.The expression of cylcin E2 was 0.83±0.04, 0.72±0.02 and 0.64±0.02 in the control group, the 20 μg/ml adiponectin group, and the 40 μg/ml adiponectin group, respectively (F=21.17, P<0.05). Conclusion Direct anti-proliferative effect has been shown in the MCF7 cells, which may be mediated by the activation of AMPK pathway and the reduction in the expression of cyclin D1 and cyclin E2. Key words: Adiponectin; Breast carcinoma; Cell cycle; Adenosine 5'-monophosphate-activated protein kinase